Microorganisms and methods for the biosynthesis of propylene

ABSTRACT

The invention provides non-naturally occurring microbial organisms having a propylene pathway. The invention additionally provides methods of using such organisms to produce propylene.

This application claims the benefit of priority of U.S. Provisionalapplication Ser. No. 61/330,258, filed Apr. 30, 2010, the entirecontents of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to biosynthetic processes, andmore specifically to organisms having propylene biosynthetic capability.

Propylene is produced primarily as a by-product of petroleum refiningand of ethylene production by steam cracking of hydrocarbon feedstocks.Propene is separated by fractional distillation from hydrocarbonmixtures obtained from cracking and other refining processes. Typicalhydrocarbon feedstocks are from non-renewable fossil fuels, such aspetroleum, natural gas and to a much lesser extent coal. Over 75 billionpounds of propylene are manufactured annually, making it the secondlargest fossil-based chemical produced behind ethylene. Propylene is abase chemical that is converted into a wide range of polymers, polymerintermediates and chemicals. Some of the most common derivatives ofchemical and polymer grade propylene are polypropylene, acrylic acid,butanol, butanediol, acrylonitrile, propylene oxide, isopropanol andcumene. The use of the propylene derivative, polypropylene, in theproduction of plastics, such as injection moulding, and fibers, such ascarpets, accounts for over one-third of U.S. consumption for thisderivative. Propylene is also used in the production of synthetic rubberand as a propellant or component in aerosols.

The ability to manufacture propylene from alternative and/or renewablefeedstocks would represent a major advance in the quest for moresustainable chemical production processes. One possible way to producepropylene renewably involves fermentation of sugars or other feedstocksto produce the alcohols 2-propanol (isopropanol) or 1-propanol, which isseparated, purified, and then dehydrated to propylene in a second stepinvolving metal-based catalysis. Direct fermentative production ofpropylene from renewable feedstocks would obviate the need fordehydration. During fermentative production, propylene gas would becontinuously emitted from the fermenter, which could be readilycollected and condensed. Developing a fermentative production processwould also eliminate the need for fossil-based propylene and would allowsubstantial savings in cost, energy, and harmful waste and emissionsrelative to petrochemically-derived propylene.

Microbial organisms and methods for effectively producing propylene fromcheap renewable feedstocks such as molasses, sugar cane juice, andsugars derived from biomass sources, including agricultural and woodwaste, as well as C1 feedstocks such as syngas and carbon dioxide, aredescribed herein and include related advantages.

SUMMARY OF THE INVENTION

The invention provides non-naturally occurring microbial organismscontaining propylene pathways comprising at least one exogenous nucleicacid encoding a propylene pathway enzyme expressed in a sufficientamount to produce propylene. The invention additionally provides methodsof using such microbial organisms to produce propylene, by culturing anon-naturally occurring microbial organism containing propylene pathwaysas described herein under conditions and for a sufficient period of timeto produce propylene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the conversion of 2-ketoglutarate to ethylene and carbondioxide by an ethylene forming enzyme.

FIG. 2 shows an exemplary pathway for production of propylene from2-ketoglutarate by a 2-ketoglutarate methyltransferase and a propyleneforming enzyme.

FIG. 3 shows exemplary pathways for production of propylene frompyruvate. Enzymes for transformation of the identified substrates toproducts include: A) a 4-hydroxy-4-methyl-2-ketoglutarate aldolase, B) a4-hydroxy-4-methyl-2-ketoglutarate dehydratase II, C) a4-hydroxy-4-methyl-2-ketoglutarate dehydratase I, D) a4-methylene-2-ketoglutarate reductase, E) a 4-methyl-2-ketoglutaconatereductase, and F) a propylene forming enzyme.

FIG. 4 shows pathways from leucine and acetoacetate to3-methyl-2-ketoglutarate. Enzymes for transformation of the identifiedsubstrates to products include: A. Leucine aminotransferase,dehydrogenase (deaminating) or oxidase, B. 4-methyl-2-oxopentanoatedehydrogenase, C. isovaleryl-CoA dehydrogenase, D. 3-methylcrotonyl-CoAcarboxylase, E. 3-methylglutaconyl-CoA hydrolase, transferase orsynthetase, F. 3-methylglutaconate hydratase, G.3-methyl-2-hydroxyglutarate dehydrogenase, H.3-hydroxy-3-methylglutaryl-CoA dehydratase and I.3-hydroxy-3-methylglutaryl-CoA lyase.

FIG. 5 shows pathways to 4-methyl-2-oxoglutarate from lysine. Enzymesfor transformation of the identified substrates to products include: A.Lysine 6-aminotransferase, 6-dehydrogenase (deaminating) or 6-oxidase,B. 2-aminoadipate dehydrogenase, C. 2-aminoadipate mutase, D.4-methyl-2-aminoglutarate aminotransferase, dehydrogenase (deaminating)or oxidase, E. 2-aminoadipate aminotransferase, dehydrogenase(deaminating) or oxidase, and F. 2-oxoadipate mutase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the design and production of cellsand organisms having biosynthetic production capabilities for propylene.The invention, in particular, relates to the design of microbialorganism capable of producing propylene by introducing one or morenucleic acids encoding a propylene pathway enzyme.

In one embodiment, the invention utilizes in silico stoichiometricmodels of Escherichia coli metabolism that identify metabolic designsfor biosynthetic production of propylene. The results described hereinindicate that metabolic pathways can be designed and recombinantlyengineered to achieve the biosynthesis of propylene in Escherichia coliand other cells or organisms. Biosynthetic production of propylene, forexample, for the in silico designs can be confirmed by construction ofstrains having the designed metabolic genotype. These metabolicallyengineered cells or organisms also can be subjected to adaptiveevolution to further augment propylene biosynthesis, including underconditions approaching theoretical maximum growth.

In certain embodiments, the propylene biosynthesis characteristics ofthe designed strains make them genetically stable and particularlyuseful in continuous bioprocesses. Separate strain design strategieswere identified with incorporation of different non-native orheterologous reaction capabilities into E. coli or other host organismsleading to propylene producing metabolic pathways from either2-ketoglutarate or pyruvate. In silico metabolic designs were identifiedthat resulted in the biosynthesis of propylene in microorganisms fromeach of these substrates or metabolic intermediates.

Strains identified via the computational component of the platform canbe put into actual production by genetically engineering any of thepredicted metabolic alterations, which lead to the biosyntheticproduction of propylene or other intermediate and/or downstreamproducts. In yet a further embodiment, strains exhibiting biosyntheticproduction of these compounds can be further subjected to adaptiveevolution to further augment product biosynthesis. The levels of productbiosynthesis yield following adaptive evolution also can be predicted bythe computational component of the system.

The maximum theoretical propylene yield from glucose is 1.33 mol/mol(0.311 g/g):3C₆H₁₂O₆=4C₃H₆+6CO₂+6H₂O

The pathways presented in FIGS. 2 and 3 achieve a yield of 1 molepropylene per mole of glucose utilized assuming 2 pyruvate molecules or1 alpha-ketoglutarate molecule can be formed from glucose. Increasingproduct yields to 1.33 mol/mol is possible if cells are capable offixing some of the CO₂ released from the depicted pathways or from theproduction of alpha-ketoglutarate through carbon-fixing mechanisms suchas the reductive (or reverse) TCA cycle or the Wood-Ljungdahl pathwaysupplemented with pyruvate:ferredoxin oxidoreductase.

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial organism's genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within a propylenebiosynthetic pathway.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides, or functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “propylene,” having the molecular formula C₃H₆and a molecular mass of 42.08 g/mol (see FIGS. 2 and 3) (IUPAC namePropene) is used interchangeably throughout with 1-propene, 1-propylene,methylethene and methylethylene. At room temperature, propylene is acolorless gas with a weak but unpleasant smell. Propylene has a higherdensity and boiling point than ethylene due to its greater size, whereasit has a slightly lower boiling point than propane in addition to beingmore volatile. Propylene lacks strongly polar bonds, yet the moleculehas a small dipole moment due to its reduced symmetry. Propylene is alsoa structural isomer to cyclopropane.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” are intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “S-Adenosyl methionine” or “SAM,” having themolecular formula C₁₅H₂₂N₆O₅S⁺ and a molecular mass of 398.44 g/mol(IUPAC name:(2S)-2-Amino-4-[[(2S,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl-methylsulfonio]butanoate)is a co-substrate involved in methyl group transfers. Transmethylation,transsulfuration, and aminopropylation are some of the metabolicpathways that use SAM. The methyl group (CH₃) attached to the methioninesulfur atom in SAM is chemically reactive. This allows donation of thisgroup to an acceptor substrate in transmethylation reactions.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

It is understood that when more than one exogenous nucleic acid isincluded in a microbial organism that the more than one exogenousnucleic acids refers to the referenced encoding nucleic acid orbiosynthetic activity, as discussed above. It is further understood, asdisclosed herein, that such more than one exogenous nucleic acids can beintroduced into the host microbial organism on separate nucleic acidmolecules, on polycistronic nucleic acid molecules, or a combinationthereof, and still be considered as more than one exogenous nucleicacid. For example, as disclosed herein a microbial organism can beengineered to express two or more exogenous nucleic acids encoding adesired pathway enzyme or protein. In the case where two exogenousnucleic acids encoding a desired activity are introduced into a hostmicrobial organism, it is understood that the two exogenous nucleicacids can be introduced as a single nucleic acid, for example, on asingle plasmid, on separate plasmids, can be integrated into the hostchromosome at a single site or multiple sites, and still be consideredas two exogenous nucleic acids. Similarly, it is understood that morethan two exogenous nucleic acids can be introduced into a host organismin any desired combination, for example, on a single plasmid, onseparate plasmids, can be integrated into the host chromosome at asingle site or multiple sites, and still be considered as two or moreexogenous nucleic acids, for example three exogenous nucleic acids.Thus, the number of referenced exogenous nucleic acids or biosyntheticactivities refers to the number of encoding nucleic acids or the numberof biosynthetic activities, not the number of separate nucleic acidsintroduced into the host organism.

The non-naturally occurring microbal organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having propylene biosyntheticcapability, those skilled in the art will understand with applying theteaching and guidance provided herein to a particular species that theidentification of metabolic modifications can include identification andinclusion or inactivation of orthologs. To the extent that paralogsand/or nonorthologous gene displacements are present in the referencedmicroorganism that encode an enzyme catalyzing a similar orsubstantially similar metabolic reaction, those skilled in the art alsocan utilize these evolutionally related genes.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism, including a microbial organism having a propylenepathway having at least one exogenous nucleic acid encoding a propylenepathway enzyme expressed in a sufficient amount to produce propylene,the propylene pathway including a 2-ketoglutarate methyltransferase or apropylene forming enzyme (see FIG. 2, steps 1-2). In one aspect, thenon-naturally occurring microbial organism includes a microbial organismhaving a propylene pathway having at least one exogenous nucleic acidencoding propylene pathway enzymes expressed in a sufficient amount toproduce propylene, the propylene pathway including a 2-ketoglutaratemethyltransferase and a propylene forming enzyme (see FIG. 2, steps 1and 2).

In some embodiments, the invention provides a non-naturally occurringmicrobial organism, including a microbial organism having a propylenepathway having at least one exogenous nucleic acid encoding a propylenepathway enzyme expressed in a sufficient amount to produce propylene,the propylene pathway including a 4-hydroxy-4-methyl-2-ketoglutaratealdolase, a 4-hydroxy-4-methyl-2-ketoglutarate dehydratase II, a4-hydroxy-4-methyl-2-ketoglutarate dehydratase I, a4-methylene-2-ketoglutarate reductase, a 4-methyl-2-ketoglutaconatereductase or a propylene forming enzyme (see FIG. 3, steps A-F). In oneaspect, the non-naturally occurring microbial organism includes amicrobial organism having a propylene pathway having at least oneexogenous nucleic acid encoding propylene pathway enzymes expressed in asufficient amount to produce propylene, the propylene pathway includinga 4-hydroxy-4-methyl-2-ketoglutarate aldolase, a4-hydroxy-4-methyl-2-ketoglutarate dehydratase II, a4-methylene-2-ketoglutarate reductase and a propylene forming enzyme(see FIG. 3, steps A, B, C and F). In one aspect, the non-naturallyoccurring microbial organism includes a microbial organism having apropylene pathway having at least one exogenous nucleic acid encodingpropylene pathway enzymes expressed in a sufficient amount to producepropylene, the propylene pathway including a4-hydroxy-4-methyl-2-ketoglutarate aldolase, a4-hydroxy-4-methyl-2-ketoglutarate dehydratase I, a4-methyl-2-ketoglutaconate reductase and a propylene forming enzyme (seeFIG. 3, steps A, D, E and F).

In some embodiments, the invention provides a non-naturally occurringmicrobial organism, including a microbial organism having a propylenepathway having at least one exogenous nucleic acid encoding a propylenepathway enzyme expressed in a sufficient amount to produce propylene,the propylene pathway including a leucine aminotransferase,dehydrogenase (deaminating) or oxidase; a 4-methyl-2-oxopentanoatedehydrogenase; an isovaleryl-CoA dehydrogenase; a 3-methylcrotonyl-CoAcarboxylase; a 3-methylglutaconyl-CoA hydrolase, transferase orsynthetase; a 3-methylglutaconate hydratase; a3-methyl-2-hydroxyglutarate dehydrogenase; a3-hydroxy-3-methylglutaryl-CoA dehydratase; or a3-hydroxy-3-methylglutaryl-CoA lyase (see FIG. 4, steps A-I). In oneaspect, the non-naturally occurring microbial organism includes amicrobial organism having a propylene pathway having at least oneexogenous nucleic acid encoding propylene pathway enzymes expressed in asufficient amount to produce propylene, the propylene pathway includinga leucine aminotransferase, dehydrogenase (deaminating) or oxidase; a4-methyl-2-oxopentanoate dehydrogenase; a isovaleryl-CoA dehydrogenase;a 3-methylcrotonyl-CoA carboxylase; a 3-methylglutaconyl-CoA hydrolase,transferase or synthetase; a 3-methylglutaconate hydratase; a3-methyl-2-hydroxyglutarate dehydrogenase and a propylene forming enzyme(see FIG. 4, steps A-G and FIG. 2, step 2). In one aspect, thenon-naturally occurring microbial organism includes a microbial organismhaving a propylene pathway having at least one exogenous nucleic acidencoding propylene pathway enzymes expressed in a sufficient amount toproduce propylene, the propylene pathway including a3-methylglutaconyl-CoA hydrolase, transferase or synthetase; a3-methylglutaconate hydratase; a 3-methyl-2-hydroxyglutaratedehydrogenase; a 3-hydroxy-3-methylglutaryl-CoA dehydratase; a3-hydroxy-3-methylglutaryl-CoA lyase and a propylene forming enzyme (seeFIG. 4, steps I, H, E-G and FIG. 2, step 2). Sources of encoding nucleicacids for a propylene pathway enzyme or protein described above are wellknown in the art and can be obtained from a variety of speciesincluding, but limited to, those exemplified herein.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism, including a microbial organism having a propylenepathway having at least one exogenous nucleic acid encoding a propylenepathway enzyme expressed in a sufficient amount to produce propylene,the propylene pathway including a lysine 6-aminotransferase, a6-dehydrogenase (deaminating) or 6-oxidase; an 2-aminoadipatedehydrogenase; an 2-aminoadipate mutase; a 4-methyl-2-aminoglutarateaminotransferase, dehydrogenase (deaminating) or oxidase; a2-aminoadipate aminotransferase, dehydrogenase (deaminating) or oxidase;or a 2-oxoadipate mutase (see FIG. 5, steps A-F). In one aspect, thenon-naturally occurring microbial organism includes a microbial organismhaving a propylene pathway having at least one exogenous nucleic acidencoding propylene pathway enzymes expressed in a sufficient amount toproduce propylene, the propylene pathway including a lysine6-aminotransferase, 6-dehydrogenase (deaminating) or 6-oxidase; an2-aminoadipate dehydrogenase, an 2-aminoadipate mutase; a4-methyl-2-aminoglutarate aminotransferase, dehydrogenase (deaminating)or oxidase; and a propylene forming enzyme (see FIG. 5, steps A-D, FIG.3, step F). In one aspect, the non-naturally occurring microbialorganism includes a microbial organism having a propylene pathway havingat least one exogenous nucleic acid encoding propylene pathway enzymesexpressed in a sufficient amount to produce propylene, the propylenepathway including a lysine 6-aminotransferase, 6-dehydrogenase(deaminating) or 6-oxidase; a 2-aminoadipate dehydrogenase; an2-aminoadipate aminotransferase, dehydrogenase (deaminating) or oxidase;an 2-oxoadipate mutase; and a propylene forming enzyme (see FIG. 5,steps A, B, E and F, FIG. 3, step F). Sources of encoding nucleic acidsfor a propylene pathway enzyme or protein described above are well knownin the art and can be obtained from a variety of species including, butlimited to, those exemplified herein.

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having a propylene pathway, wherein thenon-naturally occurring microbial organism comprises at least oneexogenous nucleic acid encoding an enzyme or protein that converts asubstrate to a product selected from the group consisting of2-ketoglutarate to 3-methyl-2-ketoglutarate, 3-methyl-2-ketoglutarate topropylene, pyruvate to 4-hydroxy-4-methyl-2-ketoglutarate,4-hydroxy-4-methyl-2-ketoglutarate to 4-methylene-2-ketoglutarate,4-methylene-2-ketoglutarate to 4-methyl-2-ketoglutarate,4-hydroxy-4-methyl-2-ketoglutarate to 4-methyl-2-ketoglutaconate,4-methyl-2-ketoglutaconate to 4-methyl-2-ketoglutarate or4-methyl-2-ketoglutarate to propylene. One skilled in the art willunderstand that these are merely exemplary and that any of thesubstrate-product pairs disclosed herein suitable to produce a desiredproduct and for which an appropriate activity is available for theconversion of the substrate to the product can be readily determined byone skilled in the art based on the teachings herein. Thus, theinvention provides a non-naturally occurring microbial organismcontaining at least one exogenous nucleic acid encoding an enzyme orprotein, where the enzyme or protein converts the substrates andproducts of a propylene pathway, such as that shown in FIGS. 2-5.

While generally described herein as a microbial organism that contains apropylene pathway, it is understood that the invention additionallyprovides a non-naturally occurring microbial organism comprising atleast one exogenous nucleic acid encoding a propylene pathway enzymeexpressed in a sufficient amount to produce an intermediate of apropylene pathway. For example, as disclosed herein, a propylene pathwayis exemplified in FIGS. 2-5. Therefore, in addition to a microbialorganism containing a propylene pathway that produces propylene, theinvention additionally provides a non-naturally occurring microbialorganism comprising at least one exogenous nucleic acid encoding apropylene pathway enzyme, where the microbial organism produces apropylene pathway intermediate, for example, 3-methyle-ketoglutarate,4-hydroxy-4-methyl-2-ketoglutarate, 4-methylene-2-ketoglutarate,4-methyl-2-ketoglutaconate, or 4-methyl-2-ketoglutarate.

It is understood that any of the pathways disclosed herein, as describedin the Examples and exemplified in the Figures, including the pathwaysof FIGS. 2-5, can be utilized to generate a non-naturally occurringmicrobial organism that produces any pathway intermediate or product, asdesired. As disclosed herein, such a microbial organism that produces anintermediate can be used in combination with another microbial organismexpressing downstream pathway enzymes to produce a desired product.However, it is understood that a non-naturally occurring microbialorganism that produces a propylene pathway intermediate can be utilizedto produce the intermediate as a desired product.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing, or a protein associated with, thereferenced metabolic reaction, reactant or product. Unless otherwiseexpressly stated herein, those skilled in the art will understand thatreference to a reaction also constitutes reference to the reactants andproducts of the reaction. Similarly, unless otherwise expressly statedherein, reference to a reactant or product also references the reaction,and reference to any of these metabolic constituents also references thegene or genes encoding the enzymes that catalyze or proteins involved inthe referenced reaction, reactant or product. Likewise, given the wellknown fields of metabolic biochemistry, enzymology and genomics,reference herein to a gene or encoding nucleic acid also constitutes areference to the corresponding encoded enzyme and the reaction itcatalyzes or a protein associated with the reaction as well as thereactants and products of the reaction.

As disclosed herein, the intermediates 3-methyl-2-ketoglutarate,4-hydroxy-4-methyl-2-ketoglutarate, 4-methyl-2-ketoglutaconate,4-methylene-2-ketoglutarate, 4-methyl-2-ketoglutarate,4-methyl-2-oxopentanoate, 3-methylglutaconate,3-methyl-2-hydroxyglutarate, 2-aminoadipate semialdehyde,2-aminoadipate, 2-oxoadipate, and 4-methyl-2-aminoglutarate, as well asother intermediates, are carboxylic acids, which can occur in variousionized forms, including fully protonated, partially protonated, andfully deprotonated forms. Accordingly, the suffix “-ate,” or the acidform, can be used interchangeably to describe both the free acid form aswell as any deprotonated form, in particular since the ionized form isknown to depend on the pH in which the compound is found. It isunderstood that carboxylate products or intermediates includes esterforms of carboxylate products or pathway intermediates, such asO-carboxylate and S-carboxylate esters. O- and S-carboxylates caninclude lower alkyl, that is C1 to C6, branched or straight chaincarboxylates. Some such O- or S-carboxylates include, withoutlimitation, methyl, ethyl, n-propyl, n-butyl, i-propyl, sec-butyl, andtert-butyl, pentyl, hexyl O- or S-carboxylates, any of which can furtherpossess an unsaturation, providing for example, propenyl, butenyl,pentyl, and hexenyl O- or S-carboxylates. O-carboxylates can be theproduct of a biosynthetic pathway. Exemplary O-carboxylates accessed viabiosynthetic pathways can include, without limitation,methyl-3-methyl-2-ketoglutarate,methyl-4-hydroxy-4-methyl-2-ketoglutarate,methyl-4-methyl-2-ketoglutaconate, methyl-4-methylene-2-ketoglutarate,methyl-4-methyl-2-ketoglutarate, methyl-4-methyl-2-oxopentanoate,methyl-3-methylglutaconate, methyl-3-methyl-2-hydroxyglutarate,methyl-2-aminoadipate semialdehyde, methyl-2-aminoadipate,methyl-2-oxoadipate, methyl-4-methyl-2-aminoglutarate,ethyl-3-methyl-2-ketoglutarate,ethyl-4-hydroxy-4-methyl-2-ketoglutarate,ethyl-4-methyl-2-ketoglutaconate, ethyl-4-methylene-2-ketoglutarate,ethyl-4-methyl-2-ketoglutarate, ethyl-4-methyl-2-oxopentanoate,ethyl-3-methylglutaconate, ethyl-3-methyl-2-hydroxyglutarate,ethyl-2-aminoadipate semialdehyde, ethyl-2-aminoadipate,ethyl-2-oxoadipate, and ethyl-4-methyl-2-aminoglutarate,n-propyl-3-methyl-2-ketoglutarate,n-propyl-4-hydroxy-4-methyl-2-ketoglutarate,n-propyl-4-methyl-2-ketoglutaconate,n-propyl-4-methylene-2-ketoglutarate, n-propyl-4-methyl-2-ketoglutarate,n-propyl-4-methyl-2-oxopentanoate, n-propyl-3-methylglutaconate,n-propyl-3-methyl-2-hydroxyglutarate, n-propyl-2-aminoadipatesemialdehyde, n-propyl-2-aminoadipate, n-propyl-2-oxoadipate, andn-propyl-4-methyl-2-aminoglutarate. Other biosynthetically accessibleO-carboxylates can include medium to long chain groups, that is C7-C22,O-carboxylate esters derived from fatty alcohols, such heptyl, octyl,nonyl, decyl, undecyl, lauryl, tridecyl, myristyl, pentadecyl, cetyl,palmitolyl, heptadecyl, stearyl, nonadecyl, arachidyl, heneicosyl, andbehenyl alcohols, any one of which can be optionally branched and/orcontain unsaturations. O-carboxylate esters can also be accessed via abiochemical or chemical process, such as esterification of a freecarboxylic acid product or transesterification of an O- orS-carboxylate. S-carboxylates are exemplified by CoA S-esters, cysteinylS-esters, alkylthioesters, and various aryl and heteroaryl thioesters.

The non-naturally occurring microbial organisms of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes or proteins participating in one or more propylenebiosynthetic pathways. Depending on the host microbial organism chosenfor biosynthesis, nucleic acids for some or all of a particularpropylene biosynthetic pathway can be expressed. For example, if achosen host is deficient in one or more enzymes or proteins for adesired biosynthetic pathway, then expressible nucleic acids for thedeficient enzyme(s) or protein(s) are introduced into the host forsubsequent exogenous expression. Alternatively, if the chosen hostexhibits endogenous expression of some pathway genes, but is deficientin others, then an encoding nucleic acid is needed for the deficientenzyme(s) or protein(s) to achieve propylene biosynthesis. Thus, anon-naturally occurring microbial organism of the invention can beproduced by introducing exogenous enzyme or protein activities to obtaina desired biosynthetic pathway or a desired biosynthetic pathway can beobtained by introducing one or more exogenous enzyme or proteinactivities that, together with one or more endogenous enzymes orproteins, produces a desired product such as propylene.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus or any of a variety of other microorganisms applicable tofermentation processes. Exemplary bacteria include species selected fromEscherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacteriumglutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcuslactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.Exemplary yeasts or fungi include species selected from Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichiapastoris, Rhizopus arrhizus, Rhizobus oryzae, Yarrowia lipolytica, andthe like. E. coli is a particularly useful host organisms since it is awell characterized microbial organism suitable for genetic engineering.Other particularly useful host organisms include yeast such asSaccharomyces cerevisiae. It is understood that any suitable microbialhost organism can be used to introduce metabolic and/or geneticmodifications to produce a desired product.

Depending on the propylene biosynthetic pathway constituents of aselected host microbial organism, the non-naturally occurring microbialorganisms of the invention will include at least one exogenouslyexpressed propylene pathway-encoding nucleic acid and up to all encodingnucleic acids for one or more propylene biosynthetic pathways. Forexample, propylene biosynthesis can be established in a host deficientin a pathway enzyme or protein through exogenous expression of thecorresponding encoding nucleic acid. In a host deficient in all enzymesor proteins of a propylene pathway, exogenous expression of all enzymeor proteins in the pathway can be included, although it is understoodthat all enzymes or proteins of a pathway can be expressed even if thehost contains at least one of the pathway enzymes or proteins. Forexample, exogenous expression of all enzymes or proteins in a pathwayfor production of propylene can be included, such as a 2-ketoglutaratemethyltransferase and a propylene forming enzyme, or alternatively, a4-hydroxy-4-methyl-2-ketoglutarate aldolase, a4-hydroxy-4-methyl-2-ketoglutarate dehydratase II, a4-methylene-2-ketoglutarate reductase and a propylene forming enzyme, oralternatively a 4-hydroxy-4-methyl-2-ketoglutarate aldolase, a4-hydroxy-4-methyl-2-ketoglutarate dehydratase I, a4-methyl-2-ketoglutaconate reductase and a propylene forming enzyme.

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel the propylenepathway deficiencies of the selected host microbial organism. Therefore,a non-naturally occurring microbial organism of the invention can haveone, two, three, or four, up to all nucleic acids encoding the enzymesor proteins constituting a propylene biosynthetic pathway disclosedherein. In some embodiments, the non-naturally occurring microbialorganisms also can include other genetic modifications that facilitateor optimize propylene biosynthesis or that confer other useful functionsonto the host microbial organism. One such other functionality caninclude, for example, augmentation of the synthesis of one or more ofthe propylene pathway precursors such as 2-ketoglutarate or pyruvate.

Generally, a host microbial organism is selected such that it producesthe precursor of a propylene pathway, either as a naturally producedmolecule or as an engineered product that either provides de novoproduction of a desired precursor or increased production of a precursornaturally produced by the host microbial organism. For example, pyruvateand 2-ketoglutarate are produced naturally in a host organism such as E.coli. A host organism can be engineered to increase production of aprecursor, as disclosed herein. In addition, a microbial organism thathas been engineered to produce a desired precursor can be used as a hostorganism and further engineered to express enzymes or proteins of apropylene pathway.

In some embodiments, a non-naturally occurring microbial organism of theinvention is generated from a host that contains the enzymaticcapability to synthesize propylene. In this specific embodiment it canbe useful to increase the synthesis or accumulation of a propylenepathway product to, for example, drive propylene pathway reactionstoward propylene production. Increased synthesis or accumulation can beaccomplished by, for example, overexpression of nucleic acids encodingone or more of the above-described propylene pathway enzymes orproteins. Overexpression of the enzyme or enzymes and/or protein orproteins of the propylene pathway can occur, for example, throughexogenous expression of the endogenous gene or genes, or throughexogenous expression of the heterologous gene or genes. Therefore,naturally occurring organisms can be readily generated to benon-naturally occurring microbial organisms of the invention, forexample, producing propylene, through overexpression of one, two, three,or four, that is, up to all nucleic acids encoding propylenebiosynthetic pathway enzymes or proteins. In addition, a non-naturallyoccurring organism can be generated by mutagenesis of an endogenous genethat results in an increase in activity of an enzyme in the propylenebiosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism.

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, a propylene biosynthetic pathway onto the microbial organism.Alternatively, encoding nucleic acids can be introduced to produce anintermediate microbial organism having the biosynthetic capability tocatalyze some of the required reactions to confer propylene biosyntheticcapability. For example, a non-naturally occurring microbial organismhaving a propylene biosynthetic pathway can comprise at least twoexogenous nucleic acids encoding desired enzymes or proteins, such asthe combination of a 2-ketoglutarate methyltransferase and a propyleneforming enzyme, or alternatively a 4-hydroxy-4-methyl-2-ketoglutaratedehydratase II and a 4-methylene-2-ketoglutarate reductase, oralternatively a 4-hydroxy-4-methyl-2-ketoglutarate aldolase and apropylene forming enzyme, and the like. Thus, it is understood that anycombination of two or more enzymes or proteins of a biosynthetic pathwaycan be included in a non-naturally occurring microbial organism of theinvention. Similarly, it is understood that any combination of three ormore enzymes or proteins of a biosynthetic pathway can be included in anon-naturally occurring microbial organism of the invention, forexample, a 4-hydroxy-4-methyl-2-ketoglutarate aldolase, a4-hydroxy-4-methyl-2-ketoglutarate dehydratase II and a4-methylene-2-ketoglutarate reductase, or alternatively a4-hydroxy-4-methyl-2-ketoglutarate dehydratase II, a4-methylene-2-ketoglutarate reductase and a propylene forming enzyme, oralternatively a 4-hydroxy-4-methyl-2-ketoglutarate aldolase, a4-hydroxy-4-methyl-2-ketoglutarate dehydratase I and a4-methyl-2-ketoglutaconate reductase, or alternatively a4-hydroxy-4-methyl-2-ketoglutarate dehydratase I, a4-methyl-2-ketoglutaconate reductase and a propylene forming enzyme andso forth, as desired, so long as the combination of enzymes and/orproteins of the desired biosynthetic pathway results in production ofthe corresponding desired product. Similarly, any combination of four, a4-hydroxy-4-methyl-2-ketoglutarate aldolase, a4-hydroxy-4-methyl-2-ketoglutarate dehydratase II, a4-methylene-2-ketoglutarate reductase and a propylene forming enzyme oralternatively a 4-hydroxy-4-methyl-2-ketoglutarate aldolase, a4-hydroxy-4-methyl-2-ketoglutarate dehydratase I, a4-methyl-2-ketoglutaconate reductase and a propylene forming enzyme, ormore enzymes or proteins of a biosynthetic pathway as disclosed hereincan be included in a non-naturally occurring microbial organism of theinvention, as desired, so long as the combination of enzymes and/orproteins of the desired biosynthetic pathway results in production ofthe corresponding desired product.

In addition to the biosynthesis of propylene as described herein, thenon-naturally occurring microbial organisms and methods of the inventionalso can be utilized in various combinations with each other and withother microbial organisms and methods well known in the art to achieveproduct biosynthesis by other routes. For example, one alternative toproduce propylene other than use of the propylene producers is throughaddition of another microbial organism capable of converting a propylenepathway intermediate to propylene. One such procedure includes, forexample, the fermentation of a microbial organism that produces apropylene pathway intermediate. The propylene pathway intermediate canthen be used as a substrate for a second microbial organism thatconverts the propylene pathway intermediate to propylene. The propylenepathway intermediate can be added directly to another culture of thesecond organism or the original culture of the propylene pathwayintermediate producers can be depleted of these microbial organisms by,for example, cell separation, and then subsequent addition of the secondorganism to the fermentation broth can be utilized to produce the finalproduct without intermediate purification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, propylene. In theseembodiments, biosynthetic pathways for a desired product of theinvention can be segregated into different microbial organisms, and thedifferent microbial organisms can be co-cultured to produce the finalproduct. In such a biosynthetic scheme, the product of one microbialorganism is the substrate for a second microbial organism until thefinal product is synthesized. For example, the biosynthesis of propylenecan be accomplished by constructing a microbial organism that containsbiosynthetic pathways for conversion of one pathway intermediate toanother pathway intermediate or the product. Alternatively, propylenealso can be biosynthetically produced from microbial organisms throughco-culture or co-fermentation using two organisms in the same vessel,where the first microbial organism produces a propylene intermediate andthe second microbial organism converts the intermediate to propylene.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methods ofthe invention together with other microbial organisms, with theco-culture of other non-naturally occurring microbial organisms havingsubpathways and with combinations of other chemical and/or biochemicalprocedures well known in the art to produce propylene.

Sources of encoding nucleic acids for a propylene pathway enzyme orprotein can include, for example, any species where the encoded geneproduct is capable of catalyzing the referenced reaction. Such speciesinclude both prokaryotic and eukaryotic organisms including, but notlimited to, bacteria, including archaea and eubacteria, and eukaryotes,including yeast, plant, insect, animal, and mammal, including human.Exemplary species for such sources include, for example, Anaerotruncuscolihominis DSM 17241, Bacteroides capillosus ATCC 29799, Campylobacterjejuni, Clostridium botulinum A3 str, Clostridium kluyveri, Clostridiumtyrobutyricum, Comamonas testosteroni, Corynebacterium glutamicum,Cupriavidus necator, Escherichia coli, Escherichia coli C, Escherichiacoli W, Eubacterium barkeri, Klebsiella pneumoniae, Methanocaldococcusjannaschii, Moorella thermoacetica, Pelotomaculum thermopropionicum,Pseudomonas ochraceae NGJ1, Pseudomonas putida F1, Pseudomonas reinekeiMT1, Pseudomonas sp. strain B13, Pseudomonas syringae pv. Glycinea,Pseudomonas syringae pv. phaseolicola PK2, Pseudomonas syringae pv.Pisi, Ralstonia eutropha JMP134, Ralstonia solanacearum, Rattusnorvegicus, Rhodococcus opacus, Saccharomyces cerevisiae, Salmonellaenterica, Sphingomonas sp. SYK6, Streptomyces coelicolor A3(2),Streptomyces fradiae, Streptomyces roseosporus NRRL 11379, Thermusthermophilus, as well as other exemplary species disclosed herein oravailable as source organisms for corresponding genes. However, with thecomplete genome sequence available for now more than 550 species (withmore than half of these available on public databases such as the NCBI),including 395 microorganism genomes and a variety of yeast, fungi,plant, and mammalian genomes, the identification of genes encoding therequisite propylene biosynthetic activity for one or more genes inrelated or distant species, including for example, homologues,orthologs, paralogs and nonorthologous gene displacements of knowngenes, and the interchange of genetic alterations between organisms isroutine and well known in the art. Accordingly, the metabolicalterations allowing biosynthesis of propylene described herein withreference to a particular organism such as E. coli can be readilyapplied to other microorganisms, including prokaryotic and eukaryoticorganisms alike. Given the teachings and guidance provided herein, thoseskilled in the art will know that a metabolic alteration exemplified inone organism can be applied equally to other organisms.

In some instances, such as when an alternative propylene biosyntheticpathway exists in an unrelated species, propylene biosynthesis can beconferred onto the host species by, for example, exogenous expression ofa paralog or paralogs from the unrelated species that catalyzes asimilar, yet non-identical metabolic reaction to replace the referencedreaction. Because certain differences among metabolic networks existbetween different organisms, those skilled in the art will understandthat the actual gene usage between different organisms may differ.However, given the teachings and guidance provided herein, those skilledin the art also will understand that the teachings and methods of theinvention can be applied to all microbial organisms using the cognatemetabolic alterations to those exemplified herein to construct amicrobial organism in a species of interest that will synthesizepropylene.

Methods for constructing and testing the expression levels of anon-naturally occurring propylene-producing host can be performed, forexample, by recombinant and detection methods well known in the art.Such methods can be found described in, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring HarborLaboratory, New York (2001); and Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for production ofpropylene can be introduced stably or transiently into a host cell usingtechniques well known in the art including, but not limited to,conjugation, electroporation, chemical transformation, transduction,transfection, and ultrasound transformation. For exogenous expression inE. coli or other prokaryotic cells, some nucleic acid sequences in thegenes or cDNAs of eukaryotic nucleic acids can encode targeting signalssuch as an N-terminal mitochondrial or other targeting signal, which canbe removed before transformation into prokaryotic host cells, ifdesired. For example, removal of a mitochondrial leader sequence led toincreased expression in E. coli (Hoffmeister et al., J. Biol. Chem.280:4329-4338 (2005)). For exogenous expression in yeast or othereukaryotic cells, genes can be expressed in the cytosol without theaddition of leader sequence, or can be targeted to mitochondrion orother organelles, or targeted for secretion, by the addition of asuitable targeting sequence such as a mitochondrial targeting orsecretion signal suitable for the host cells. Thus, it is understoodthat appropriate modifications to a nucleic acid sequence to remove orinclude a targeting sequence can be incorporated into an exogenousnucleic acid sequence to impart desirable properties. Furthermore, genescan be subjected to codon optimization with techniques well known in theart to achieve optimized expression of the proteins.

An expression vector or vectors can be constructed to include one ormore propylene biosynthetic pathway encoding nucleic acids asexemplified herein operably linked to expression control sequencesfunctional in the host organism. Expression vectors applicable for usein the microbial host organisms of the invention include, for example,plasmids, phage vectors, viral vectors, episomes and artificialchromosomes, including vectors and selection sequences or markersoperable for stable integration into a host chromosome. Additionally,the expression vectors can include one or more selectable marker genesand appropriate expression control sequences. Selectable marker genesalso can be included that, for example, provide resistance toantibiotics or toxins, complement auxotrophic deficiencies, or supplycritical nutrients not in the culture media. Expression controlsequences can include constitutive and inducible promoters,transcription enhancers, transcription terminators, and the like whichare well known in the art. When two or more exogenous encoding nucleicacids are to be co-expressed, both nucleic acids can be inserted, forexample, into a single expression vector or in separate expressionvectors. For single vector expression, the encoding nucleic acids can beoperationally linked to one common expression control sequence or linkedto different expression control sequences, such as one induciblepromoter and one constitutive promoter. The transformation of exogenousnucleic acid sequences involved in a metabolic or synthetic pathway canbe confirmed using methods well known in the art. Such methods include,for example, nucleic acid analysis such as Northern blots or polymerasechain reaction (PCR) amplification of mRNA, or immunoblotting forexpression of gene products, or other suitable analytical methods totest the expression of an introduced nucleic acid sequence or itscorresponding gene product. It is understood by those skilled in the artthat the exogenous nucleic acid is expressed in a sufficient amount toproduce the desired product, and it is further understood thatexpression levels can be optimized to obtain sufficient expression usingmethods well known in the art and as disclosed herein.

In some embodiments, the invention provides a method for producingpropylene that includes culturing a non-naturally occurring microbialorganism, including a microbial organism having a propylene pathway, thepropylene pathway including at least one exogenous nucleic acid encodinga propylene pathway enzyme expressed in a sufficient amount to producepropylene, the propylene pathway including a 2-ketoglutaratemethyltransferase or a propylene forming enzyme (see FIG. 2, steps 1-2).In one aspect, the method includes a microbial organism having apropylene pathway including a 2-ketoglutarate methyltransferase and apropylene forming enzyme (FIG. 2, steps 1 and 2).

In some embodiments, the invention provides a method for producingpropylene that includes culturing a non-naturally occurring microbialorganism, including a microbial organism having a propylene pathway, thepropylene pathway including at least one exogenous nucleic acid encodinga propylene pathway enzyme expressed in a sufficient amount to producepropylene, the propylene pathway including a4-hydroxy-4-methyl-2-ketoglutarate aldolase, a4-hydroxy-4-methyl-2-ketoglutarate dehydratase II, a4-hydroxy-4-methyl-2-ketoglutarate dehydratase I, a4-methylene-2-ketoglutarate reductase, a 4-methyl-2-ketoglutaconatereductase or a propylene forming enzyme (see FIG. 3, steps A-F). In oneaspect, the method includes a microbial organism having a propylenepathway including a 4-hydroxy-4-methyl-2-ketoglutarate aldolase, a4-hydroxy-4-methyl-2-ketoglutarate dehydratase II, a4-methylene-2-ketoglutarate reductase and a propylene forming enzyme(see FIG. 3, steps A, B, C and F). In one aspect, the method includes amicrobial organism having a propylene pathway including a4-hydroxy-4-methyl-2-ketoglutarate aldolase, a4-hydroxy-4-methyl-2-ketoglutarate dehydratase I, a4-methyl-2-ketoglutaconate reductase and a propylene forming enzyme(FIG. 3, steps A, D, E and F).

In some embodiments, the invention provides a method for producingpropylene that includes culturing a non-naturally occurring microbialorganism, including a microbial organism having a propylene pathway, thepropylene pathway including at least one exogenous nucleic acid encodinga propylene pathway enzyme expressed in a sufficient amount to producepropylene, the propylene pathway including a leucine aminotransferase,dehydrogenase (deaminating) or oxidase; a 4-methyl-2-oxopentanoatedehydrogenase; an isovaleryl-CoA dehydrogenase; a 3-methylcrotonyl-CoAcarboxylase; a 3-methylglutaconyl-CoA hydrolase, transferase orsynthetase; a 3-methylglutaconate hydratase; a3-methyl-2-hydroxyglutarate dehydrogenase; a3-hydroxy-3-methylglutaryl-CoA dehydratase; or a3-hydroxy-3-methylglutaryl-CoA lyase (see FIG. 4, steps A-I). In oneaspect, the method includes a microbial organism having a propylenepathway including a leucine aminotransferase, dehydrogenase(deaminating) or oxidase; a 4-methyl-2-oxopentanoate dehydrogenase; aisovaleryl-CoA dehydrogenase; a 3-methylcrotonyl-CoA carboxylase; a3-methylglutaconyl-CoA hydrolase, transferase or synthetase; a3-methylglutaconate hydratase; a 3-methyl-2-hydroxyglutaratedehydrogenase and a propylene forming enzyme (see FIG. 4, steps A-G andFIG. 2, step 2). In one aspect, the method includes a microbial organismhaving a propylene pathway including a 3-methylglutaconyl-CoA hydrolase,transferase or synthetase; a 3-methylglutaconate hydratase; a3-methyl-2-hydroxyglutarate dehydrogenase; a3-hydroxy-3-methylglutaryl-CoA dehydratase; a3-hydroxy-3-methylglutaryl-CoA lyase and a propylene forming enzyme (seeFIG. 4, steps I, H, E-G and FIG. 2, step 2). Sources of encoding nucleicacids for a propylene pathway enzyme or protein described above are wellknown in the art and can be obtained from a variety of speciesincluding, but limited to, those exemplified herein.

In some embodiments, the invention provides a method for producingpropylene that includes culturing a non-naturally occurring microbialorganism, including a microbial organism having a propylene pathway, thepropylene pathway including at least one exogenous nucleic acid encodinga propylene pathway enzyme expressed in a sufficient amount to producepropylene, the propylene pathway including a lysine 6-aminotransferase,a 6-dehydrogenase (deaminating) or 6-oxidase; an 2-aminoadipatedehydrogenase; an 2-aminoadipate mutase; a 4-methyl-2-aminoglutarateaminotransferase, dehydrogenase (deaminating) or oxidase; a2-aminoadipate aminotransferase, dehydrogenase (deaminating) or oxidase;or a 2-oxoadipate mutase (see FIG. 5, steps A-F). In one aspect, themethod includes a microbial organism having a propylene pathwayincluding a lysine 6-aminotransferase, 6-dehydrogenase (deaminating) or6-oxidase; an 2-aminoadipate dehydrogenase, an 2-aminoadipate mutase; a4-methyl-2-aminoglutarate aminotransferase, dehydrogenase (deaminating)or oxidase; and a propylene forming enzyme (see FIG. 5, steps A-D, FIG.3, step F). In one aspect, the method includes a microbial organismhaving a propylene pathway including a lysine 6-aminotransferase,6-dehydrogenase (deaminating) or 6-oxidase; a 2-aminoadipatedehydrogenase; an 2-aminoadipate aminotransferase, dehydrogenase(deaminating) or oxidase; an 2-oxoadipate mutase; and a propyleneforming enzyme (see FIG. 5, steps A, B, E and F, FIG. 3, step F).Sources of encoding nucleic acids for a propylene pathway enzyme orprotein described above are well known in the art and can be obtainedfrom a variety of species including, but limited to, those exemplifiedherein.

Suitable purification and/or assays to test for the production ofpropylene can be performed using well known methods. Suitable replicatessuch as triplicate cultures can be grown for each engineered strain tobe tested. For example, product and byproduct formation in theengineered production host can be monitored. The final product andintermediates, and other organic compounds, can be analyzed by methodssuch as HPLC (High Performance Liquid Chromatography), GC-MS (GasChromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-MassSpectroscopy) or other suitable analytical methods using routineprocedures well known in the art. The release of product in thefermentation broth can also be tested with the culture supernatant.Byproducts and residual glucose can be quantified by HPLC using, forexample, a refractive index detector for glucose and alcohols, and a UVdetector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779(2005)), or other suitable assay and detection methods well known in theart. The individual enzyme or protein activities from the exogenous DNAsequences can also be assayed using methods well known in the art. Fortypical Assay Methods, see Manual on Hydrocarbon Analysis (ASTM ManulaSeries, A. W. Drews, ed., 6^(th) edition, 1998, American Society forTesting and Materials, Baltimore, Md.

The propylene can be separated from other components in the cultureusing a variety of methods well known in the art. Such separationmethods include, for example, extraction procedures as well as methodsthat include continuous liquid-liquid extraction, pervaporation,membrane filtration, membrane separation, reverse osmosis,electrodialysis, distillation, crystallization, centrifugation,extractive filtration, ion exchange chromatography, size exclusionchromatography, adsorption chromatography, and ultrafiltration. All ofthe above methods are well known in the art.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the propylene producers can be cultured forthe biosynthetic production of propylene.

For the production of propylene, the recombinant strains are cultured ina medium with carbon source and other essential nutrients. It issometimes desirable and can be highly desirable to maintain anaerobicconditions in the fermenter to reduce the cost of the overall process.Such conditions can be obtained, for example, by first sparging themedium with nitrogen and then sealing the flasks with a septum andcrimp-cap. For strains where growth is not observed anaerobically,microaerobic or substantially anaerobic conditions can be applied byperforating the septum with a small hole for limited aeration. Exemplaryanaerobic conditions have been described previously and are well-knownin the art. Exemplary aerobic and anaerobic conditions are described,for example, in United State publication 2009/0047719, filed Aug. 10,2007. Fermentations can be performed in a batch, fed-batch or continuousmanner, as disclosed herein.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

The growth medium can include, for example, any carbohydrate sourcewhich can supply a source of carbon to the non-naturally occurringmicroorganism. Such sources include, for example, sugars such asglucose, xylose, arabinose, galactose, mannose, fructose, sucrose andstarch. Other sources of carbohydrate include, for example, renewablefeedstocks and biomass. Exemplary types of biomasses that can be used asfeedstocks in the methods of the invention include cellulosic biomass,hemicellulosic biomass and lignin feedstocks or portions of feedstocks.Such biomass feedstocks contain, for example, carbohydrate substratesuseful as carbon sources such as glucose, xylose, arabinose, galactose,mannose, fructose and starch. Given the teachings and guidance providedherein, those skilled in the art will understand that renewablefeedstocks and biomass other than those exemplified above also can beused for culturing the microbial organisms of the invention for theproduction of propylene.

In addition to renewable feedstocks such as those exemplified above, thepropylene microbial organisms of the invention also can be modified forgrowth on syngas as its source of carbon. In this specific embodiment,one or more proteins or enzymes are expressed in the propylene producingorganisms to provide a metabolic pathway for utilization of syngas orother gaseous carbon source.

Synthesis gas, also known as syngas or producer gas, is the majorproduct of gasification of coal and of carbonaceous materials such asbiomass materials, including agricultural crops and residues. Syngas isa mixture primarily of H₂ and CO and can be obtained from thegasification of any organic feedstock, including but not limited tocoal, coal oil, natural gas, biomass, and waste organic matter.Gasification is generally carried out under a high fuel to oxygen ratio.Although largely H₂ and CO, syngas can also include CO₂ and other gasesin smaller quantities. Thus, synthesis gas provides a cost effectivesource of gaseous carbon such as CO and, additionally, CO₂.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ toacetyl-CoA and other products such as acetate. Organisms capable ofutilizing CO and syngas also generally have the capability of utilizingCO₂ and CO₂/H₂ mixtures through the same basic set of enzymes andtransformations encompassed by the Wood-Ljungdahl pathway. H₂-dependentconversion of CO₂ to acetate by microorganisms was recognized longbefore it was revealed that CO also could be used by the same organismsand that the same pathways were involved. Many acetogens have been shownto grow in the presence of CO₂ and produce compounds such as acetate aslong as hydrogen is present to supply the necessary reducing equivalents(see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, NewYork, (1994)). This can be summarized by the following equation:2CO₂+4H₂ +nADP+nPi→CH₃COOH+2H₂O+nATP

Hence, non-naturally occurring microorganisms possessing theWood-Ljungdahl pathway can utilize CO₂ and H₂ mixtures as well for theproduction of acetyl-CoA and other desired products.

The Wood-Ljungdahl pathway is well known in the art and consists of 12reactions which can be separated into two branches: (1) methyl branchand (2) carbonyl branch. The methyl branch converts syngas tomethyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branchconverts methyl-THF to acetyl-CoA. The reactions in the methyl branchare catalyzed in order by the following enzymes or proteins: ferredoxinoxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase. The reactions in the carbonyl branch are catalyzed in orderby the following enzymes or proteins: methyltetrahydrofolate:corrinoidprotein methyltransferase (for example, AcsE), corrinoid iron-sulfurprotein, nickel-protein assembly protein (for example, AcsF),ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase andnickel-protein assembly protein (for example, CooC). Following theteachings and guidance provided herein for introducing a sufficientnumber of encoding nucleic acids to generate a propylene pathway, thoseskilled in the art will understand that the same engineering design alsocan be performed with respect to introducing at least the nucleic acidsencoding the Wood-Ljungdahl enzymes or proteins absent in the hostorganism. Therefore, introduction of one or more encoding nucleic acidsinto the microbial organisms of the invention such that the modifiedorganism contains the complete Wood-Ljungdahl pathway will confer syngasutilization ability.

Additionally, the reductive (reverse) tricarboxylic acid cycle coupledwith carbon monoxide dehydrogenase and/or hydrogenase activities canalso be used for the conversion of CO, CO₂ and/or H₂ to acetyl-CoA andother products such as acetate. Organisms capable of fixing carbon viathe reductive TCA pathway can utilize one or more of the followingenzymes: ATP citrate-lyase, citrate lyase, aconitase, isocitratedehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase,succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase,fumarase, malate dehydrogenase, NAD(P)H:ferredoxin oxidoreductase,carbon monoxide dehydrogenase, and hydrogenase. Specifically, thereducing equivalents extracted from CO and/or H₂ by carbon monoxidedehydrogenase and hydrogenase are utilized to fix CO₂ via the reductiveTCA cycle into acetyl-CoA or acetate. Acetate can be converted toacetyl-CoA by enzymes such as acetyl-CoA transferase, acetatekinase/phosphotransacetylase, and acetyl-CoA synthetase. Acetyl-CoA canbe converted to the p-toluate, terepathalate, or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate precursors,glyceraldehyde-3-phosphate, phosphoenolpyruvate, and pyruvate, bypyruvate:ferredoxin oxidoreductase and the enzymes of gluconeogenesis.Following the teachings and guidance provided herein for introducing asufficient number of encoding nucleic acids to generate a p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway,those skilled in the art will understand that the same engineeringdesign also can be performed with respect to introducing at least thenucleic acids encoding the reductive TCA pathway enzymes or proteinsabsent in the host organism. Therefore, introduction of one or moreencoding nucleic acids into the microbial organisms of the inventionsuch that the modified organism contains the complete reductive TCApathway will confer syngas utilization ability.

Accordingly, given the teachings and guidance provided herein, thoseskilled in the art will understand that a non-naturally occurringmicrobial organism can be produced that secretes the biosynthesizedcompounds of the invention when grown on a carbon source such as acarbohydrate. Such compounds include, for example, propylene and any ofthe intermediate metabolites in the propylene pathway. All that isrequired is to engineer in one or more of the required enzyme or proteinactivities to achieve biosynthesis of the desired compound orintermediate including, for example, inclusion of some or all of thepropylene biosynthetic pathways. Accordingly, the invention provides anon-naturally occurring microbial organism that produces and/or secretespropylene when grown on a carbohydrate or other carbon source andproduces and/or secretes any of the intermediate metabolites shown inthe propylene pathway when grown on a carbohydrate or other carbonsource. The propylene producing microbial organisms of the invention caninitiate synthesis from an intermediate, for example,3-methyle-ketoglutarate, 4-hydroxy-4-methyl-2-ketoglutarate,4-methylene-2-ketoglutarate, 4-methyl-2-ketoglutaconate, or4-methyl-2-ketoglutarate.

The non-naturally occurring microbial organisms of the invention areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding a propylenepathway enzyme or protein in sufficient amounts to produce propylene. Itis understood that the microbial organisms of the invention are culturedunder conditions sufficient to produce propylene. Following theteachings and guidance provided herein, the non-naturally occurringmicrobial organisms of the invention can achieve biosynthesis ofpropylene resulting in intracellular concentrations between about0.001-2000 mM or more. Generally, the intracellular concentration ofpropylene is between about 3-1500 mM, particularly between about 5-1250mM and more particularly between about 8-1000 mM, including about 100mM, 200 mM, 500 mM, 800 mM, or more. Intracellular concentrationsbetween and above each of these exemplary ranges also can be achievedfrom the non-naturally occurring microbial organisms of the invention.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. publication2009/0047719, filed Aug. 10, 2007. Any of these conditions can beemployed with the non-naturally occurring microbial organisms as well asother anaerobic conditions well known in the art. Under such anaerobicor substantially anaerobic conditions, the propylene producers cansynthesize propylene at intracellular concentrations of 5-10 mM or moreas well as all other concentrations exemplified herein. It is understoodthat, even though the above description refers to intracellularconcentrations, propylene producing microbial organisms can producepropylene intracellularly and/or secrete the product into the culturemedium.

In addition to the culturing and fermentation conditions disclosedherein, growth condition for achieving biosynthesis of propylene caninclude the addition of an osmoprotectant to the culturing conditions.In certain embodiments, the non-naturally occurring microbial organismsof the invention can be sustained, cultured or fermented as describedherein in the presence of an osmoprotectant. Briefly, an osmoprotectantrefers to a compound that acts as an osmolyte and helps a microbialorganism as described herein survive osmotic stress. Osmoprotectantsinclude, but are not limited to, betaines, amino acids, and the sugartrehalose. Non-limiting examples of such are glycine betaine, pralinebetaine, dimethylthetin, dimethylslfonioproprionate,3-dimethylsulfonio-2-methylproprionate, pipecolic acid,dimethylsulfonioacetate, choline, L-carnitine and ectoine. In oneaspect, the osmoprotectant is glycine betaine. It is understood to oneof ordinary skill in the art that the amount and type of osmoprotectantsuitable for protecting a microbial organism described herein fromosmotic stress will depend on the microbial organism used. The amount ofosmoprotectant in the culturing conditions can be, for example, no morethan about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM,no more than about 1.5 mM, no more than about 2.0 mM, no more than about2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no morethan about 7.0 mM, no more than about 10 mM, no more than about 50 mM,no more than about 100 mM or no more than about 500 mM.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of propylene includes anaerobic culture or fermentationconditions. In certain embodiments, the non-naturally occurringmicrobial organisms of the invention can be sustained, cultured orfermented under anaerobic or substantially anaerobic conditions.Briefly, anaerobic conditions refers to an environment devoid of oxygen.Substantially anaerobic conditions include, for example, a culture,batch fermentation or continuous fermentation such that the dissolvedoxygen concentration in the medium remains between 0 and 10% ofsaturation. Substantially anaerobic conditions also includes growing orresting cells in liquid medium or on solid agar inside a sealed chambermaintained with an atmosphere of less than 1% oxygen. The percent ofoxygen can be maintained by, for example, sparging the culture with anN₂/CO₂ mixture or other suitable non-oxygen gas or gases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of propylene. Exemplary growth proceduresinclude, for example, fed-batch fermentation and batch separation;fed-batch fermentation and continuous separation, or continuousfermentation and continuous separation. All of these processes are wellknown in the art. Fermentation procedures are particularly useful forthe biosynthetic production of commercial quantities of propylene.Generally, and as with non-continuous culture procedures, the continuousand/or near-continuous production of propylene will include culturing anon-naturally occurring propylene producing organism of the invention insufficient nutrients and medium to sustain and/or nearly sustain growthin an exponential phase. Continuous culture under such conditions caninclude, for example, growth for 1 day, 2, 3, 4, 5, 6 or 7 days or more.Additionally, continuous culture can include longer time periods of 1week, 2, 3, 4 or 5 or more weeks and up to several months.Alternatively, organisms of the invention can be cultured for hours, ifsuitable for a particular application. It is to be understood that thecontinuous and/or near-continuous culture conditions also can includeall time intervals in between these exemplary periods. It is furtherunderstood that the time of culturing the microbial organism of theinvention is for a sufficient period of time to produce a sufficientamount of product for a desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of propylene can be utilized in, forexample, fed-batch fermentation and batch separation; fed-batchfermentation and continuous separation, or continuous fermentation andcontinuous separation. Examples of batch and continuous fermentationprocedures are well known in the art.

In addition to the above fermentation procedures using the propyleneproducers of the invention for continuous production of substantialquantities of propylene, the propylene producers also can be, forexample, simultaneously subjected to chemical synthesis procedures toconvert the product to other compounds or the product can be separatedfrom the fermentation culture and sequentially subjected to chemical orenzymatic conversion to convert the product to other compounds, ifdesired.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of propylene.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion or disruption strategies that result ingenetically stable microorganisms which overproduce the target product.Specifically, the framework examines the complete metabolic and/orbiochemical network of a microorganism in order to suggest geneticmanipulations that force the desired biochemical to become an obligatorybyproduct of cell growth. By coupling biochemical production with cellgrowth through strategically placed gene deletions or other functionalgene disruption, the growth selection pressures imposed on theengineered strains after long periods of time in a bioreactor lead toimprovements in performance as a result of the compulsory growth-coupledbiochemical production. Lastly, when gene deletions are constructedthere is a negligible possibility of the designed strains reverting totheir wild-type states because the genes selected by OptKnock are to becompletely removed from the genome. Therefore, this computationalmethodology can be used to either identify alternative pathways thatlead to biosynthesis of a desired product or used in connection with thenon-naturally occurring microbial organisms for further optimization ofbiosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat allow an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. publication 2002/0168654, filed Jan. 10, 2002, in InternationalPatent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. publication2009/0047719, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

As disclosed herein, a nucleic acid encoding a desired activity of apropylene pathway can be introduced into a host organism. In some cases,it can be desirable to modify an activity of a propylene pathway enzymeor protein to increase production of propylene. For example, knownmutations that increase the activity of a protein or enzyme can beintroduced into an encoding nucleic acid molecule. Additionally,optimization methods can be applied to increase the activity of anenzyme or protein and/or decrease an inhibitory activity, for example,decrease the activity of a negative regulator.

One such optimization method is directed evolution. Directed evolutionis a powerful approach that involves the introduction of mutationstargeted to a specific gene in order to improve and/or alter theproperties of an enzyme. Improved and/or altered enzymes can beidentified through the development and implementation of sensitivehigh-throughput screening assays that allow the automated screening ofmany enzyme variants (for example, >10⁴). Iterative rounds ofmutagenesis and screening typically are performed to afford an enzymewith optimized properties. Computational algorithms that can help toidentify areas of the gene for mutagenesis also have been developed andcan significantly reduce the number of enzyme variants that need to begenerated and screened. Numerous directed evolution technologies havebeen developed (for reviews, see Hibbert et al., Biomol. Eng 22:11-19(2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical andbiotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press;Otten and Quax. Biomol. Eng 22:1-9 (2005).; and Sen et al., ApplBiochem. Biotechnol 143:212-223 (2007)) to be effective at creatingdiverse variant libraries, and these methods have been successfullyapplied to the improvement of a wide range of properties across manyenzyme classes. Enzyme characteristics that have been improved and/oraltered by directed evolution technologies include, for example:selectivity/specificity, for conversion of non-natural substrates;temperature stability, for robust high temperature processing; pHstability, for bioprocessing under lower or higher pH conditions;substrate or product tolerance, so that high product titers can beachieved; binding (K_(m)), including broadening substrate binding toinclude non-natural substrates; inhibition (K_(i)), to remove inhibitionby products, substrates, or key intermediates; activity (kcat), toincreases enzymatic reaction rates to achieve desired flux; expressionlevels, to increase protein yields and overall pathway flux; oxygenstability, for operation of air sensitive enzymes under aerobicconditions; and anaerobic activity, for operation of an aerobic enzymein the absence of oxygen.

Described below in more detail are exemplary methods that have beendeveloped for the mutagenesis and diversification of genes to targetdesired properties of specific enzymes. Such methods are well known tothose skilled in the art. Any of these can be used to alter and/oroptimize the activity of a propylene pathway enzyme or protein.

EpPCR (Pritchard et al., J Theor. Biol. 234:497-509 (2005)) introducesrandom point mutations by reducing the fidelity of DNA polymerase in PCRreactions by the addition of Mn²⁺ ions, by biasing dNTP concentrations,or by other conditional variations. The five step cloning process toconfine the mutagenesis to the target gene of interest involves: 1)error-prone PCR amplification of the gene of interest; 2) restrictionenzyme digestion; 3) gel purification of the desired DNA fragment; 4)ligation into a vector; 5) transformation of the gene variants into asuitable host and screening of the library for improved performance.This method can generate multiple mutations in a single genesimultaneously, which can be useful to screen a larger number ofpotential variants having a desired activity. A high number of mutantscan be generated by EpPCR, so a high-throughput screening assay or aselection method, for example, using robotics, is useful to identifythose with desirable characteristics.

Error-prone Rolling Circle Amplification (epRCA) (Fujii et al., NucleicAcids Res. 32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497(2006)) has many of the same elements as epPCR except a whole circularplasmid is used as the template and random 6-mers with exonucleaseresistant thiophosphate linkages on the last 2 nucleotides are used toamplify the plasmid followed by transformation into cells in which theplasmid is re-circularized at tandem repeats. Adjusting the Mn²⁺concentration can vary the mutation rate somewhat. This technique uses asimple error-prone, single-step method to create a full copy of theplasmid with 3-4 mutations/kbp. No restriction enzyme digestion orspecific primers are required. Additionally, this method is typicallyavailable as a commercially available kit.

DNA or Family Shuffling (Stemmer, Proc Natl Acad Sci USA 91:10747-10751(1994)); and Stemmer, Nature 370:389-391 (1994)) typically involvesdigestion of two or more variant genes with nucleases such as Dnase I orEndoV to generate a pool of random fragments that are reassembled bycycles of annealing and extension in the presence of DNA polymerase tocreate a library of chimeric genes. Fragments prime each other andrecombination occurs when one copy primes another copy (templateswitch). This method can be used with >1 kbp DNA sequences. In additionto mutational recombinants created by fragment reassembly, this methodintroduces point mutations in the extension steps at a rate similar toerror-prone PCR. The method can be used to remove deleterious, randomand neutral mutations.

Staggered Extension (StEP) (Zhao et al., Nat. Biotechnol. 16:258-261(1998)) entails template priming followed by repeated cycles of 2 stepPCR with denaturation and very short duration of annealing/extension (asshort as 5 sec). Growing fragments anneal to different templates andextend further, which is repeated until full-length sequences are made.Template switching means most resulting fragments have multiple parents.Combinations of low-fidelity polymerases (Taq and Mutazyme) reduceerror-prone biases because of opposite mutational spectra.

In Random Priming Recombination (RPR) random sequence primers are usedto generate many short DNA fragments complementary to different segmentsof the template (Shao et al., Nucleic Acids Res 26:681-683 (1998)). Basemisincorporation and mispriming via epPCR give point mutations. ShortDNA fragments prime one another based on homology and are recombined andreassembled into full-length by repeated thermocycling. Removal oftemplates prior to this step assures low parental recombinants. Thismethod, like most others, can be performed over multiple iterations toevolve distinct properties. This technology avoids sequence bias, isindependent of gene length, and requires very little parent DNA for theapplication.

In Heteroduplex Recombination linearized plasmid DNA is used to formheteroduplexes that are repaired by mismatch repair (Volkov et al,Nucleic Acids Res. 27:e18 (1999); and Volkov et al., Methods Enzymol.328:456-463 (2000)). The mismatch repair step is at least somewhatmutagenic. Heteroduplexes transform more efficiently than linearhomoduplexes. This method is suitable for large genes and whole operons.

Random Chimeragenesis on Transient Templates (RACHITT) (Coco et al.,Nat. Biotechnol. 19:354-359 (2001)) employs Dnase I fragmentation andsize fractionation of single stranded DNA (ssDNA). Homologous fragmentsare hybridized in the absence of polymerase to a complementary ssDNAscaffold. Any overlapping unhybridized fragment ends are trimmed down byan exonuclease. Gaps between fragments are filled in and then ligated togive a pool of full-length diverse strands hybridized to the scaffold,which contains U to preclude amplification. The scaffold then isdestroyed and is replaced by a new strand complementary to the diversestrand by PCR amplification. The method involves one strand (scaffold)that is from only one parent while the priming fragments derive fromother genes; the parent scaffold is selected against. Thus, noreannealing with parental fragments occurs. Overlapping fragments aretrimmed with an exonuclease. Otherwise, this is conceptually similar toDNA shuffling and StEP. Therefore, there should be no siblings, fewinactives, and no unshuffled parentals. This technique has advantages inthat few or no parental genes are created and many more crossovers canresult relative to standard DNA shuffling.

Recombined Extension on Truncated templates (RETT) entails templateswitching of unidirectionally growing strands from primers in thepresence of unidirectional ssDNA fragments used as a pool of templates(Lee et al., J. Molec. Catalysis 26:119-129 (2003)). No DNAendonucleases are used. Unidirectional ssDNA is made by DNA polymerasewith random primers or serial deletion with exonuclease. UnidirectionalssDNA are only templates and not primers. Random priming andexonucleases do not introduce sequence bias as true of enzymaticcleavage of DNA shuffling/RACHITT. RETT can be easier to optimize thanStEP because it uses normal PCR conditions instead of very shortextensions. Recombination occurs as a component of the PCR steps, thatis, no direct shuffling. This method can also be more random than StEPdue to the absence of pauses.

In Degenerate Oligonucleotide Gene Shuffling (DOGS) degenerate primersare used to control recombination between molecules; (Bergquist andGibbs, Methods Mol. Biol. 352:191-204 (2007); Bergquist et al., Biomol.Eng 22:63-72 (2005); Gibbs et al., Gene 271:13-20 (2001)) this can beused to control the tendency of other methods such as DNA shuffling toregenerate parental genes. This method can be combined with randommutagenesis (epPCR) of selected gene segments. This can be a good methodto block the reformation of parental sequences. No endonucleases areneeded. By adjusting input concentrations of segments made, one can biastowards a desired backbone. This method allows DNA shuffling fromunrelated parents without restriction enzyme digests and allows a choiceof random mutagenesis methods.

Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY)creates a combinatorial library with 1 base pair deletions of a gene orgene fragment of interest (Ostermeier et al., Proc. Natl. Acad. Sci. USA96:3562-3567 (1999); and Ostermeier et al., Nat. Biotechnol.17:1205-1209 (1999)). Truncations are introduced in opposite directionon pieces of 2 different genes. These are ligated together and thefusions are cloned. This technique does not require homology between the2 parental genes. When ITCHY is combined with DNA shuffling, the systemis called SCRATCHY (see below). A major advantage of both is no need forhomology between parental genes; for example, functional fusions betweenan E. coli and a human gene were created via ITCHY. When ITCHY librariesare made, all possible crossovers are captured.

Thio-Incremental Truncation for the Creation of Hybrid Enzymes(THIO-ITCHY) is similar to ITCHY except that phosphothioate dNTPs areused to generate truncations (Lutz et al., Nucleic Acids Res 29:E16(2001)). Relative to ITCHY, THIO-ITCHY can be easier to optimize,provide more reproducibility, and adjustability.

SCRATCHY combines two methods for recombining genes, ITCHY and DNAshuffling (Lutz et al., Proc. Natl. Acad. Sci. USA 98:11248-11253(2001)). SCRATCHY combines the best features of ITCHY and DNA shuffling.First, ITCHY is used to create a comprehensive set of fusions betweenfragments of genes in a DNA homology-independent fashion. Thisartificial family is then subjected to a DNA-shuffling step to augmentthe number of crossovers. Computational predictions can be used inoptimization. SCRATCHY is more effective than DNA shuffling whensequence identity is below 80%.

In Random Drift Mutagenesis (RNDM) mutations made via epPCR followed byscreening/selection for those retaining usable activity (Bergquist etal., Biomol. Eng. 22:63-72 (2005)). Then, these are used in DOGS togenerate recombinants with fusions between multiple active mutants orbetween active mutants and some other desirable parent. Designed topromote isolation of neutral mutations; its purpose is to screen forretained catalytic activity whether or not this activity is higher orlower than in the original gene. RNDM is usable in high throughputassays when screening is capable of detecting activity above background.RNDM has been used as a front end to DOGS in generating diversity. Thetechnique imposes a requirement for activity prior to shuffling or othersubsequent steps; neutral drift libraries are indicated to result inhigher/quicker improvements in activity from smaller libraries. Thoughpublished using epPCR, this could be applied to other large-scalemutagenesis methods.

Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis methodthat: 1) generates a pool of random length fragments using randomincorporation of a phosphothioate nucleotide and cleavage; this pool isused as a template to 2) extend in the presence of “universal” basessuch as inosine; 3) replication of an inosine-containing complementgives random base incorporation and, consequently, mutagenesis (Wong etal., Biotechnol. J. 3:74-82 (2008); Wong et al., Nucleic Acids Res.32:e26 (2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)).Using this technique it can be possible to generate a large library ofmutants within 2 to 3 days using simple methods. This technique isnon-directed in comparison to the mutational bias of DNA polymerases.Differences in this approach makes this technique complementary (or analternative) to epPCR.

In Synthetic Shuffling, overlapping oligonucleotides are designed toencode “all genetic diversity in targets” and allow a very highdiversity for the shuffled progeny (Ness et al., Nat. Biotechnol.20:1251-1255 (2002)). In this technique, one can design the fragments tobe shuffled. This aids in increasing the resulting diversity of theprogeny. One can design sequence/codon biases to make more distantlyrelated sequences recombine at rates approaching those observed withmore closely related sequences. Additionally, the technique does notrequire physically possessing the template genes.

Nucleotide Exchange and Excision Technology NexT exploits a combinationof dUTP incorporation followed by treatment with uracil DNA glycosylaseand then piperidine to perform endpoint DNA fragmentation (Muller etal., Nucleic Acids Res. 33:e117 (2005)). The gene is reassembled usinginternal PCR primer extension with proofreading polymerase. The sizesfor shuffling are directly controllable using varying dUPT::dTTP ratios.This is an end point reaction using simple methods for uracilincorporation and cleavage. Other nucleotide analogs, such as8-oxo-guanine, can be used with this method. Additionally, the techniqueworks well with very short fragments (86 bp) and has a low error rate.The chemical cleavage of DNA used in this technique results in very fewunshuffled clones.

In Sequence Homology-Independent Protein Recombination (SHIPREC), alinker is used to facilitate fusion between two distantly related orunrelated genes. Nuclease treatment is used to generate a range ofchimeras between the two genes. These fusions result in libraries ofsingle-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460(2001)). This produces a limited type of shuffling and a separateprocess is required for mutagenesis. In addition, since no homology isneeded, this technique can create a library of chimeras with varyingfractions of each of the two unrelated parent genes. SHIPREC was testedwith a heme-binding domain of a bacterial CP450 fused to N-terminalregions of a mammalian CP450; this produced mammalian activity in a moresoluble enzyme.

In Gene Site Saturation Mutagenesis™ (GSSM™) the starting materials area supercoiled dsDNA plasmid containing an insert and two primers whichare degenerate at the desired site of mutations (Kretz et al., MethodsEnzymol. 388:3-11 (2004)). Primers carrying the mutation of interest,anneal to the same sequence on opposite strands of DNA. The mutation istypically in the middle of the primer and flanked on each side byapproximately 20 nucleotides of correct sequence. The sequence in theprimer is NNN or NNK (coding) and MNN (noncoding) (N=all 4, K=G, T, M=A,C). After extension, DpnI is used to digest dam-methylated DNA toeliminate the wild-type template. This technique explores all possibleamino acid substitutions at a given locus (that is, one codon). Thetechnique facilitates the generation of all possible replacements at asingle-site with no nonsense codons and results in equal to near-equalrepresentation of most possible alleles. This technique does not requireprior knowledge of the structure, mechanism, or domains of the targetenzyme. If followed by shuffling or Gene Reassembly, this technologycreates a diverse library of recombinants containing all possiblecombinations of single-site up-mutations. The usefulness of thistechnology combination has been demonstrated for the successfulevolution of over 50 different enzymes, and also for more than oneproperty in a given enzyme.

Combinatorial Cassette Mutagenesis (CCM) involves the use of shortoligonucleotide cassettes to replace limited regions with a large numberof possible amino acid sequence alterations (Reidhaar-Olson et al.Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al. Science241:53-57 (1988)). Simultaneous substitutions at two or three sites arepossible using this technique. Additionally, the method tests a largemultiplicity of possible sequence changes at a limited range of sites.This technique has been used to explore the information content of thelambda repressor DNA-binding domain.

Combinatorial Multiple Cassette Mutagenesis (CMCM) is essentiallysimilar to CCM except it is employed as part of a larger program: 1) useof epPCR at high mutation rate to 2) identify hot spots and hot regionsand then 3) extension by CMCM to cover a defined region of proteinsequence space (Reetz et al., Angew. Chem. Int. Ed Engl. 40:3589-3591(2001)). As with CCM, this method can test virtually all possiblealterations over a target region. If used along with methods to createrandom mutations and shuffled genes, it provides an excellent means ofgenerating diverse, shuffled proteins. This approach was successful inincreasing, by 51-fold, the enantioselectivity of an enzyme.

In the Mutator Strains technique, conditional ts mutator plasmids allowincreases of 20 to 4000-X in random and natural mutation frequencyduring selection and block accumulation of deleterious mutations whenselection is not required (Selifonova et al., Appl. Environ. Microbiol.67:3645-3649 (2001)). This technology is based on a plasmid-derivedmutD5 gene, which encodes a mutant subunit of DNA polymerase III. Thissubunit binds to endogenous DNA polymerase III and compromises theproofreading ability of polymerase III in any strain that harbors theplasmid. A broad-spectrum of base substitutions and frameshift mutationsoccur. In order for effective use, the mutator plasmid should be removedonce the desired phenotype is achieved; this is accomplished through atemperature sensitive (ts) origin of replication, which allows forplasmid curing at 41° C. It should be noted that mutator strains havebeen explored for quite some time (see Low et al., J. Mol. Biol.260:359-3680 (1996)). In this technique, very high spontaneous mutationrates are observed. The conditional property minimizes non-desiredbackground mutations. This technology could be combined with adaptiveevolution to enhance mutagenesis rates and more rapidly achieve desiredphenotypes.

Look-Through Mutagenesis (LTM) is a multidimensional mutagenesis methodthat assesses and optimizes combinatorial mutations of selected aminoacids (Rajpal et al., Proc. Natl. Acad. Sci. USA 102:8466-8471 (2005)).Rather than saturating each site with all possible amino acid changes, aset of nine is chosen to cover the range of amino acid R-groupchemistry. Fewer changes per site allows multiple sites to be subjectedto this type of mutagenesis. A >800-fold increase in binding affinityfor an antibody from low nanomolar to picomolar has been achievedthrough this method. This is a rational approach to minimize the numberof random combinations and can increase the ability to find improvedtraits by greatly decreasing the numbers of clones to be screened. Thishas been applied to antibody engineering, specifically to increase thebinding affinity and/or reduce dissociation. The technique can becombined with either screens or selections.

Gene Reassembly is a DNA shuffling method that can be applied tomultiple genes at one time or to create a large library of chimeras(multiple mutations) of a single gene (Tunable GeneReassembly™ (TGR™)Technology supplied by Verenium Corporation). Typically this technologyis used in combination with ultra-high-throughput screening to query therepresented sequence space for desired improvements. This techniqueallows multiple gene recombination independent of homology. The exactnumber and position of cross-over events can be pre-determined usingfragments designed via bioinformatic analysis. This technology leads toa very high level of diversity with virtually no parental genereformation and a low level of inactive genes. Combined with GSSM™, alarge range of mutations can be tested for improved activity. The methodallows “blending” and “fine tuning” of DNA shuffling, for example, codonusage can be optimized.

In Silico Protein Design Automation (PDA) is an optimization algorithmthat anchors the structurally defined protein backbone possessing aparticular fold, and searches sequence space for amino acidsubstitutions that can stabilize the fold and overall protein energetics(Hayes et al., Proc. Natl. Acad. Sci. USA 99:15926-15931 (2002)). Thistechnology uses in silico structure-based entropy predictions in orderto search for structural tolerance toward protein amino acid variations.Statistical mechanics is applied to calculate coupling interactions ateach position. Structural tolerance toward amino acid substitution is ameasure of coupling. Ultimately, this technology is designed to yielddesired modifications of protein properties while maintaining theintegrity of structural characteristics. The method computationallyassesses and allows filtering of a very large number of possiblesequence variants (10⁵⁰). The choice of sequence variants to test isrelated to predictions based on the most favorable thermodynamics.Ostensibly only stability or properties that are linked to stability canbe effectively addressed with this technology. The method has beensuccessfully used in some therapeutic proteins, especially inengineering immunoglobulins. In silico predictions avoid testingextraordinarily large numbers of potential variants. Predictions basedon existing three-dimensional structures are more likely to succeed thanpredictions based on hypothetical structures. This technology canreadily predict and allow targeted screening of multiple simultaneousmutations, something not possible with purely experimental technologiesdue to exponential increases in numbers.

Iterative Saturation Mutagenesis (ISM) involves: 1) using knowledge ofstructure/function to choose a likely site for enzyme improvement; 2)performing saturation mutagenesis at chosen site using a mutagenesismethod such as Stratagene QuikChange (Stratagene; San Diego Calif.); 3)screening/selecting for desired properties; and 4) using improvedclone(s), start over at another site and continue repeating until adesired activity is achieved (Reetz et al., Nat. Protoc. 2:891-903(2007); and Reetz et al., Angew. Chem. Int. Ed Engl. 45:7745-7751(2006)). This is a proven methodology, which assures all possiblereplacements at a given position are made for screening/selection.

Any of the aforementioned methods for mutagenesis can be used alone orin any combination. Additionally, any one or combination of the directedevolution methods can be used in conjunction with adaptive evolutiontechniques, as described herein.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Example I Pathways for Producing Propylene from 2-Ketoglutarate orPyruvate

Disclosed herein are novel processes for the direct production ofpropylene using engineered non-natural microorganisms that possess theenzymes necessary for conversion of common metabolites into the threecarbon alkene, propylene. Direct production of propylene entails use ofthe well-known ethylene forming enzyme (EFE), which has been isolatedand characterized from, for example, pathovars of the plant pathogenPseudomonas syringae and shown to convert 2-ketoglutarate into ethyleneand carbon dioxide (FIG. 1). The EFE enzyme or a variant of EFE is usedfor conversion of 3-methyl-2-ketoglutarate or 4-methyl-2-ketoglutarateinto propylene and carbon dioxide in a manner analogous to theconversion of 2-ketoglutarate (step 2 of FIG. 2 and step F of FIG. 3).Thus we refer this enzyme herein as propylene forming enzyme (PFE). Theintermediate 3-methyl-2-ketoglutarate can be formed directly from2-ketoglutarate through an S-adenosylmethionine-dependentmethyltransferase enzyme such as that encoded by, for example, the geneglmT in Streptomyces frudiae and other organisms (step 1 of FIG. 2).Alternatively, it can be formed from leucine or acetoacetate andacetyl-CoA as depicted in FIG. 4. The intermediate4-methyl-2-oxo-glutarate can be formed through aldolase-catalyzedtransformation of pyruvate to 4-hydroxy-4-methyl-2-ketoglutarate,followed by dehydration with the enzyme or variant of malate dehydrataseand subsequent reduction with an enoate reductase or with an enzyme orvariant of fumarate reductase (steps A-E of FIG. 3). Alternatively, itcan be formed from lysine depicted in FIG. 5. Enzyme candidates forsteps 1 and 2 of FIG. 2 and steps A-F of FIG. 3 are provided below.

Step 1 of FIG. 2 depicts 2-ketoglutarate methyltransferase whichcatalyzes the methylation of 2-ketoglutarate to form3-methyl-2-ketoglutarate. Such activity can be obtained using enzymesencoded by glmT from Streptomyces coelicolor, dptI from Streptomycesroseosporus, and lptI from Streptomyces fradiae (Mahlert et al., J. Am.Chem. Soc., 2007, 129 (39), 12011-12018).

GenBank GI Gene Accession No. Number Organism glmT NP_627429.1 21221650Streptomyces coelicolor A3(2) dptI ZP_04706744.1 239986080 Streptomycesroseosporus NRRL 11379 lptI AAZ23087.1 71068232 Streptomyces fradiae

Several candidate genes are likely to naturally exhibit PFE activity oneither 3-methyl-2-ketoglutarate (step 2, FIG. 2) or4-methyl-2-ketoglutarate (step F, FIG. 3). If not, they can beengineered to exhibit PFE activity on these substrates. Candidate genesinclude EFEs from various strains of Pseudomonas syringae and Ralstoniasolanacearum (Fukuda et al., Biochem Biophys Res Comm, 1992, 188 (2),826-832; Tao et al., Appl Microbiol Biotechnol, 2008, 80(4):573-8; Chenet al., Int J of Biol Sci, 2010, 6(1):96-106; Weingart et al.,Phytopathology, 1999, 89:360-365).

GenBank GI Gene Accession No. Number Organism Efe BAA02477.1 216878Pseudomonas syringae pv. phaseolicola PK2 Efe AAD16443.1 4323603Pseudomonas syringae pv. Pisi Efe Q7BS32.1 50897474 Pseudomonas syringaepv. glycinea Efe CAD18680.1 17432003 Ralstonia solanacearum

Step A of FIG. 3 depicts 4-hydroxy-4-methyl-2-ketoglutarate aldolasewhich catalyzes the condensation of two pyruvate molecules to form4-hydroxy-4-methyl-2-ketoglutarate. Genes from the following organismsencoded enzymes that catalyze 4-hydroxy-4-methyl-2-ketoglutaratealdolase: Pseudomonas ochraceae NGJ1 (Maruyama et al., Biosci BiotechnolBiochem, 2001, 65(12):2701-2709), Pseudomonas putida (Dagley, MethodsEnzymol, 1982, 90:272-276), and Pseudomonas testosteroni (or Comamonastestosteroni) (Providenti et al., Microbiology, 2001 147 (Pt 8),2157-2167), and Arachis hypogaea.

GenBank GI Gene Accession No. Number Organism proA BAB21456.3 13094205Pseudomonas ochraceae NGJ1 Pput_1361 ABQ77519.1 148510659 Pseudomonasputida F1 Pput_3204 ABQ79330.1 148512470 Pseudomonas putida F1CtCNB1_2744 YP_003278786.1 264678879 Comamonas testosteroni

Step D of FIG. 3 depicts 4-hydroxy-4-methyl-2-ketoglutarate dehydrataseI which dehydrates 4-hydroxy-4-methyl-2-ketoglutarate to form4-methyl-2-ketoglutaconate. Step B of FIG. 3 depicts4-hydroxy-4-methyl-2-ketoglutarate dehydratase II which dehydrates4-hydroxy-4-methyl-2-ketoglutarate to form 4-methylene-2-ketoglutarate.Various classes of dehydratases can be applied to catalyze thesetransformations. Specific examples are provided below and severaladditional dehydratase enzymes in the EC 4.2.1 can be alternativelyutilized.

For example, one enzyme that catalyzes a similar transformation iscitramalate hydrolyase (EC 4.2.1.34), an enzyme that naturallydehydrates 2-methylmalate to mesaconate. This enzyme has been studied inMethanocaldococcus jannaschii in the context of the pyruvate pathway to2-oxobutanoate, where it has been shown to have a broad substratespecificity (Drevland et al., J. Bacteriol. 189:4391-4400 (2007)). Thisenzyme activity was also detected in Clostridium tetanomorphum,Morganella morganii, Citrobacter amalonaticus where it is thought toparticipate in glutamate degradation (Kato and Asano Arch. Microbiol.168:457-463 (1997)). The M. jannaschii protein sequence does not bearsignificant homology to genes in these organisms. Genbank informationrelated to this gene is summarized below.

GenBank GI Gene Accession No. Number Organism leuD 3122345 Q58673.1Methanocaldococcus jannaschii

Another useful enzyme is fumarate hydratase (EC 4.2.1.2), also known asfumarase, that catalyzes the reversible hydration of fumarate to malate.The three fumarases of E. coli, encoded by fumA, fumB and fumC, areregulated under different conditions of oxygen availability. FumB isoxygen sensitive and is active under anaerobic conditions. FumA isactive under microanaerobic conditions, and FumC is active under aerobicgrowth conditions (Tseng et al., J. Bacteriol. 183:461-467 (2001); Woodset al., Biochim. Biophys. Acta 954:14-26 (1988); Guest et al., J. Gen.Microbiol. 131:2971-2984 (1985)). S. cerevisiae contains one copy of afumarase-encoding gene, FUM1, whose product localizes to both thecytosol and mitochondrion (Sass et al., J. Biol. Chem. 278:45109-45116(2003)). Additional fumarase enzymes are found in Campylobacter jejuni(Smith et al., Int. J. Biochem. Cell. Biol. 31:961-975 (1999)), Thermusthermophilus (Mizobata et al., Arch. Biochem. Biophys. 355:49-55 (1998))and Rattus norvegicus (Kobayashi et al., J. Biochem. 89:1923-1931(1981)). Similar enzymes with high sequence homology include fum1 fromArabidopsis thaliana and fumC from Corynebacterium glutamicum. The MmcBCfumarase from Pelotomaculum thermopropionicum is another class offumarase with two subunits (Shimoyama et al., FEMS Microbiol. Lett.270:207-213 (2007)). Genbank information related to these genes issummarized below.

GenBank GI Gene Accession No. Number Organism fumA NP_416129.1 16129570Escherichia coli fumB NP_418546.1 16131948 Escherichia coli fumCNP_416128.1 16129569 Escherichia coli FUM1 NP_015061 6324993Saccharomyces cerevisiae fumC Q8NRN8.1 39931596 Corynebacteriumglutamicum fumC O69294.1 9789756 Campylobacter jejuni fumC P8412775427690 Thermus thermophilus fumH P14408.1 120605 Rattus norvegicusMmcB YP_001211906 147677691 Pelotomaculum thermopropionicum MmcCYP_001211907 147677692 Pelotomaculum thermopropionicum

The enzyme OHED hydratase participates in 4-hydroxyphenylacetic aciddegradation, where it converts 2-oxo-hept-4-ene-1,7-dioate (OHED) to2-oxo-4-hydroxy-hepta-1,7-dioate (HODH) using magnesium as a cofactor(Burks et al., J. Am. Chem. Soc. 120 (1998). OHED hydratase enzymes havebeen identified and characterized in E. coli C (Izumi et al., J. Mol.Biol. 370:899-911 (2007); Roper et al., Gene 156:47-51 (1995)) and E.coli W (Prieto et al., J. Bacteriol. 178:111-120 (1996)). Sequencecomparison reveals homologs in a range of bacteria, plants and animals.Enzymes with highly similar sequences are contained in Klebsiellapneumonia (91% identity, evalue=2e-138) and Salmonella enterica (91%identity, evalue=4e-138), among others. Genbank information related tothese genes is summarized below.

GenBank GI Gene Accession No. Number Organism hpcG CAA57202.1 556840Escherichia coli C hpaH CAA86044.1 757830 Escherichia coli W hpaHABR80130.1 150958100 Klebsiella pneumoniae Sari_01896 ABX21779.1160865156 Salmonella enterica

Yet another enzyme catalyzing a dehydration similar to step D of FIG. 3is 2-(hydroxymethyl)glutarate dehydratase of Eubacterium barkeri. Thisenzyme has been studied in the context of nicotinate catabolism and isencoded by hmd (Alhapel et al., Proc. Natl. Acad. Sci. USA103:12341-12346 (2006)). Similar enzymes with high sequence homology arefound in Bacteroides capillosus and Anaerotruncus colihominis. Theseenzymes are also homologous to the α- and β-subunits of[4Fe-4S]-containing bacterial serine dehydratases, for example, E. colienzymes encoded by tdcG, sdhB, and sdaA). Genbank information related tothese genes is summarized below.

GenBank GI Gene Accession No. Number Organism hmd ABC88407.1 86278275Eubacterium barkeri BACCAP_02294 ZP_02036683.1 154498305 Bacteroidescapillosus ATCC 29799 ANACOL_02527 ZP_02443222.1 167771169 Anaerotruncuscolihominis DSM 17241

Another gene capable of encoding a dehydratase enzyme for step B or stepD of FIG. 3 is 4-oxalomesaconate hydratase (or4-carboxy-4-hydroxy-2-ketoadipate dehydratase). Exemplary enzymes can befound in Comamonas testosteroni (Providenti et al., Microbiology, 2001,147 (Pt 8); 2157-67), Sphingomonas sp. SYK6 (Hara, et al., J Bacteriol,2000, 182(24); 6950-7), and Pseudomonas ochraceae NGJ1 (Maruyama et al.,Biosci Biotechnol Biochem, 2001 65(12); 2701-9; Maruyama et al., BiosciBiotechnol Biochem, 2004, 68(7); 1434-41).

GenBank GI Gene Accession No. Number Organism pmdE YP_003278787.1264678880 Comamonas testosteroni ligJ BAA97116.1 8777583 Sphingomonassp. SYK6 proH BAB21455.1 12539404 Pseudomonas ochraceae NGJ1

Step C of FIG. 3 depicts 4-methylene-2-ketoglutarate reductase whichreduces 4-methylene-2-ketoglutarate reductase to form4-methyl-2-ketoglutarate. Step E of FIG. 3 depicts4-methyl-2-ketoglutaconate reductase which reduces4-methyl-2-ketoglutaconate to form 4-methyl-2-ketoglutarate. Variousclasses of enoate reductases can be applied to catalyze thesetransformations. Specific examples are provided below and severaladditional enoate reductase enzymes in the EC 1.3.1 can be alternativelyutilized.

2-Enoate oxidoreductase enzymes are known to catalyze theNAD(P)H-dependent reduction and oxidation of a wide variety ofα,β-unsaturated carboxylic acids and aldehydes (Rohdich et al., J. Biol.Chem. 276:5779-5787 (2001)). In the recently published genome sequenceof C. kluyveri, 9 coding sequences for enoate reductases were reported,out of which one has been characterized (Seedorf et al., Proc. Natl.Acad. Sci. U.S.A. 105:2128-2133 (2008)). The enr genes from both C.tyrobutyricum and M. thermoaceticum have been cloned and sequenced andshow 59% identity to each other. The former gene is also found to haveapproximately 75% similarity to the characterized gene in C. kluyveri(Giesel and Simon, Arch. Microbiol. 135:51-57 (1983)). It has beenreported based on these sequence results that enr is very similar to thedienoyl CoA reductase in E. coli (fadH) (Rohdich et al., J. Biol. Chem.276:5779-5787 (2001)). The C. thermoaceticum enr gene has also beenexpressed in a catalytically active form in E. coli (Rohdich et al.,supra). Genbank information related to these genes is summarized below.

GenBank GI Gene Accession No. Number Organism enr ACA54153.1 169405742Clostridium botulinum A3 str enr CAA71086.1 2765041 Clostridiumtyrobutyricum enr CAA76083.1 3402834 Clostridium kluyveri enrYP_430895.1 83590886 Moorella thermoacetica fadH NP_417552.1 16130976Escherichia coli

MAR is a 2-enoate oxidoreductase catalyzing the reversible reduction of2-maleylacetate (cis-4-oxohex-2-enedioate) to 3-oxoadipate (FIG. 2, StepO). MAR enzymes naturally participate in aromatic degradation pathways(Camara et al., J. Bacteriol. 191:4905-4915 (2009); Huang et al., Appl.Environ. Microbiol. 72:7238-7245 (2006); Kaschabek and Reineke, J.Bacteriol. 177:320-325 (1995); Kaschabek and Reineke, J. Bacteriol.175:6075-6081 (1993)). The enzyme activity was identified andcharacterized in Pseudomonas sp. strain B13 (Kaschabek and Reineke,(1995) supra; Kaschabek and Reineke, (1993) supra), and the coding genewas cloned and sequenced (Kasberg et al., J. Bacteriol. 179:3801-3803(1997)). Additional MAR genes include cicE gene from Pseudomonas sp.strain B13 (Kasberg et al., supra), macA gene from Rhodococcus opacus(Seibert et al., J. Bacteriol. 175:6745-6754 (1993)), the macA gene fromRalstonia eutropha (also known as Cupriavidus necator) (Seibert et al.,Microbiology 150:463-472 (2004)), tfdFII from Ralstonia eutropha(Seibert et al., (1993) supra) and NCgl1112 in Corynebacteriumglutamicum (Huang et al., Appl. Environ Microbiol. 72:7238-7245 (2006)).A MAR in Pseudomonas reinekei MT1, encoded by ccaD, was recentlyidentified and the nucleotide sequence is available under the DBJ/EMBLGenBank accession number EF159980 (Camara et al., J. Bacteriol.191:4905-4915 (2009). Genbank information related to these genes issummarized below.

GenBank GI Gene Accession No. Number Organism clcE O30847.1 3913241Pseudomonas sp. strain B13 macA O84992.1 7387876 Rhodococcus opacus macAAAD55886 5916089 Cupriavidus necator tfdFII AC44727.1 1747424 Ralstoniaeutropha JMP134 NCgl1112 NP_600385 19552383 Corynebacterium glutamicumccaD EF159980.1 134133935 Pseudomonas reinekei MT1

Fumarate reductase catalyzes the reduction of fumarate to succinate. Thefumarate reductase of E. coli, composed of four subunits encoded byfrdABCD, is membrane-bound and active under anaerobic conditions. Theelectron donor for this reaction is menaquinone and the two protonsproduced in this reaction do not contribute to the proton gradient(Iverson et al., Science 284:1961-1966 (1999)). The yeast genome encodestwo soluble fumarate reductase isozymes encoded by FRDS1 (Enomoto etal., DNA Res. 3:263-267 (1996)) and FRDS2 (Muratsubaki et al., Arch.Biochem. Biophys. 352:175-181 (1998)), which localize to the cytosol andpromitochondrion, respectively, and are used for anaerobic growth onglucose (Arikawa et al., FEMS Microbiol. Lett. 165:111-116 (1998)).

GenBank GI Protein Accession No. Number Organism FRDS1 P32614 418423Saccharomyces cerevisiae FRDS2 NP_012585 6322511 Saccharomycescerevisiae frdA NP_418578.1 16131979 Escherichia coli frdB NP_418577.116131978 Escherichia coli frdC NP_418576.1 16131977 Escherichia colifrdD NP_418475.1 16131877 Escherichia coli

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties, includingGenBank and GI number publications, are hereby incorporated by referencein this application in order to more fully describe the state of the artto which this invention pertains. Although the invention has beendescribed with reference to the examples provided above, it should beunderstood that various modifications can be made without departing fromthe spirit of the invention.

What is claimed is:
 1. A non-naturally occurring microbial organismhaving a propylene pathway and comprising at least two exogenous nucleicacids encoding propylene pathway enzymes expressed in a sufficientamount to produce propylene, wherein said propylene pathway comprises anenzyme that converts 2-ketoglutarate to 3-methyl-2-ketoglutarate and anenzyme that converts 3-methyl-2-ketoglutarate to propylene, wherein saidenzyme that converts 2-ketoglutarate to 3-methyl-2-ketoglutarate is amethyltransferase, and wherein said enzyme that converts3-methyl-2-ketoglutarate to propylene is an ethylene forming enzyme,wherein the organism produces propylene.
 2. The non-naturally occurringmicrobial organism of claim 1, wherein said at least two exogenousnucleic acids are heterologous nucleic acids.
 3. The non-naturallyoccurring microbial organism of claim 1, wherein said non-naturallyoccurring microbial organism is in a substantially anaerobic culturemedium.
 4. The non-naturally occurring microbial organism of claim 1,wherein said ethylene forming enzyme is selected from the organismsPseudomonas syringae pv. phaseolicola PK2, Pseudomonas syringae pv.Pisi, Pseudomonas syringae pv. glycinea and Ralstonia solanacearum. 5.The non-naturally occurring microbial organism of claim 1, wherein saidmethyltransferase is selected from the organisms Streptomycescoelicolor, Streptomyces roseosporus and Streptomyces fradiae.
 6. Amedium comprising: a non-naturally occurring microbial organism having apropylene pathway and comprising at least two exogenous nucleic acidsencoding propylene pathway enzymes expressed in a sufficient amount toproduce propylene, wherein said propylene pathway comprises an enzymethat converts 2-ketoglutarate to 3-methyl-2-ketoglutarate and an enzymethat converts 3-methyl-2-ketoglutarate to propylene, wherein said enzymethat converts 2-ketoglutarate to 3-methyl-2-ketoglutarate is amethyltransferase, and wherein said enzyme that converts3-methyl-2-ketoglutarate to propylene is an ethylene forming enzyme; andpropylene.
 7. A method for producing propylene, comprising culturing thenon-naturally occurring microbial organism of claim 1 under conditionsand for a sufficient period of time to produce propylene and separatingthe propylene from other components in the culture.
 8. The method ofclaim 7, wherein said non-naturally occurring microbial organism is in asubstantially anaerobic culture medium.
 9. The non-naturally occurringmicrobial organism of claim 1, wherein said wherein said microbialorganism is a species of bacteria, yeast, or fungus.
 10. The method ofclaim 7, wherein the separating comprises extraction, continuousliquid-liquid extraction, pervaporation, membrane filtration, membraneseparation, reverse osmosis, electrodialysis, distillation,crystallization, centrifugation, extractive filtration, ion exchangechromatography, absorption chromatography, or ultrafiltration.